Cancer treatment and immune system regulation through FAT10 pathway inhibition

ABSTRACT

Described herein are methods of inhibiting mitosis, treating cancer and/or treating immune disorders through the use of agents that inhibit FAT10 and/or the FAT10 pathway.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/390,265, filed Oct. 2, 2014, which is the national stage filing under35 U.S.C. § 371 of PCT/US2013/034950, filed Apr. 2, 2013, which claimsthe benefit of U.S. Provisional Application No. 61/619,091, filed Apr.2, 2012, the content of which is expressly incorporated by referenceherein in its entirety.

GOVERNMENT INTEREST

This invention was made with Government support under NationalInstitutes of Health Grant RO1 GM039023. The Government has certainrights in the invention.

BACKGROUND

The number of different proteins and protein isoforms in the humanproteome is estimated to be about three orders of magnitude higher thanthe number of genes encoded in the genome. This diversity is largely dueto post-translational modification of proteins, Such modifications canhave a significant impact on protein function and stability.

The Ubiquitin-Like (Ubl) molecule family play a prominent role inpost-transcriptional modification-based protein regulation. The Ublmolecule family are a class of evolutionarily conserved polypeptidesthat can be reversibly conjugated to lysine residues on the proteinsthey regulate through the formation of isopeptide bonds. The binding ofa Ubl to a protein can affect the protein's activity, stability,cellular localization and/or its interaction with other proteins. SomeUbl conjugation pathways are known to be important in various humandiseases, including in cancer, viral infection and neurodegenerativedisorders. More than a dozen Ubl family members have been identified todate.

FAT10 is a Ubl that is homologous to di-ubiquitin and has been suggestedto be the only Ubl modifier that targets proteins for degradationthrough conjugation. However, the only in vivo covalent substrates ofFAT10 identified to date are Ube2z and p53. It is therefore unclearwhether FAT10 acts as part of a signaling pathway or acts to funnelproteins to the proteasome for degradation. The role of FAT10 in humandisease remains unknown.

SUMMARY

Provided herein are compositions and methods for the inhibition ofmitosis, the inhibition of cellular proliferation, the induction ofapoptosis, the treatment of cancer, the treatment of immune disordersand/or the identification of novel therapeutic agents.

In some embodiments, provided herein are methods for inhibiting mitosis,cellular proliferation and/or the induction of apoptosis in a cell. Insome embodiments the methods described herein include contacting thecell with an agent that inhibits the FAT10 pathway in the cell (e.g., acancer cell or an immune cell). In some embodiments the agent inhibitsthe FAT10 pathway by inhibiting the activity or expression of FAT10 inthe cell. In some embodiments the cell is in a subject who has or issuspected of having cancer or an immune disorder. In some embodiments,the cell is subject to conditions that induce FAT10 expression, such asin the presence of pro-inflammatory cytokines.

In certain embodiments, provided herein are methods of treating canceror an immune disorder in a subject. In some embodiments, the methodscomprise the steps of administering to the subject an agent thatinhibits the FAT10 pathway. In some embodiments the agent inhibits theFAT10 pathway by inhibiting the activity or expression of FAT10. In someembodiments the cancer is melanoma.

In certain embodiments of the methods described herein, the agent can bea small molecule, a polypeptide and/or an inhibitory nucleic acid. Forexample, the agent could be a small molecule that inhibits FAT10activity or an inhibitory nucleic acid (e.g., siRNA, shRNA, antisenseRNA) that is specific for FAT10 mRNA. In some embodiments the agentinhibits the formation of a conjugate between FAT10 and a FAT10substrate (e.g., a FAT10 substrate encoded by a nucleic acid provided inFIG. 20).

In some embodiments, provided herein are methods for determining whethera test agent is a candidate therapeutic agent for treating cancer and/orimmune disorders.

In some embodiments the method includes the steps of: a) forming a testreaction mixture comprising a FAT10 protein, a FAT10 substrate, aconcentrated mammalian cell extract or a tissue sample (e.g., a tumorsample) and a test agent, b) incubating the test reaction underconditions conducive for the formation of a conjugate between the FAT10protein and the FAT10 substrate (e.g., a FAT10 substrate encoded by anucleic acid sequence provided in FIG. 20), and c) determining theamount of the conjugate in the test reaction mixture. In general, a testagent that reduces the amount of the conjugate in the test reactionmixture compared to the amount of the conjugate in a control reactionmixture is a candidate therapeutic agent for the treatment of cancerand/or immune disorders. The control reaction mixture can be, forexample, a reaction mixture that is substantially identical to the testreaction mixture except that the control reaction mixture does notcomprise a test agent or a reaction mixture that is substantiallyidentical to the test reaction mixture except that the control reactionmixture comprises a placebo agent instead of a test agent. In someembodiments, the FAT10 protein and/or the FAT10 substrate is linked(directly or indirectly) to a detectable moiety. In some embodiments,the FAT10 protein and/or the FAT10 substrate is anchored (eitherdirectly or indirectly) to a solid support. In some embodiments, theFAT10/FAT10 substrate conjugate is isolated from unconjugated FAT10protein and/or unconjugated FAT10 substrate.

In some embodiments the method includes the steps of: a) contacting acell, a cell extract or a tissue sample (e.g., a tumor sample)with thetest agent, and b) detecting the expression or activity of FAT10 in thecell, cell extract or tissue sample. In general, a test agent thatdecreases the expression or activity of FAT10 in a cell is a candidatetherapeutic agent for treating cancer and/or immune disorders. In someembodiments, expression of FAT10 is detected in a cell by detectingFAT10 mRNA level or FAT10 protein level in the cell. In someembodiments, the activity of FAT10 is detected in a cell, cell extractor tissue sample by detecting a conjugate that includes FAT10 and aFAT10 substrate (e.g., a FAT10 substrate encoded by a nucleic acidprovided in FIG. 20).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of selecting the set of “reactive proteins” forubiquitin. Signal intensity values (α-poly ubiquitin antibody: y-axis)were plotted against the RFU values (relative abundance on the chip;x-axis) for each spot. The RFU range was then divided into 100equal-sized bins and the mean and SD intensity values for the spots ineach bin were calculated, Based on the complementary error function eachprotein was assigned a p-value (FDR corrected) and proteins that hadp<0.05 were selected (orange: p<0.05; blue: p>=0.05). Note that they-axis for signal intensity is on a log scale.

FIG. 2 shows the experimental design for the Ubl modification assay.Mitotic HeLa S3 cell extract were incubated on protein microarrays withor without the addition of Ubch10, a protein that abrogates thecheckpoint arrest and allows the extracts to proceed toward mitoticexit. Ubl modifications on the spotted proteins are then measured bylabeling the arrays with UBL-specific antibodies, andfluorescently-labeled secondary antibodies are used to quantify thereactivity profile of the ˜8000 proteins on the array toward each Ublmodification.

FIG. 3 shows a list of the protein targets (by official gene symbol)that passed the reactivity threshold for each of the Ubl modifiers. Eachcolumn represents one Ubl. A shaded box indicates an interaction betweenthe protein and the Ubl was detected, while a white box indicates thatno interaction between the protein and Ubl was detected.

FIG. 4A shows examples of known Ubl targets and Ubl pathway enzymesidentified by the assay. A grey box denotes reactivity toward that Ubl,while a black box indicates no interaction. FIG. 4B shows the Ublinteraction network. Each protein is connected to the Ubl with which itinteracts. Proteins that have multiple Ubls interactions are shown atthe center and proteins that are reactive exclusively with one Ubl areshown at the rim. FIG. 4C shows the number of proteins targeted by eachUbl showing specificity of the Ubl pathways.

FIG. 5 shows a comparison of the observed distribution of Ublreactivities with expected distribution for a random Ubl network

FIG. 6 shows an enrichment analyses of “molecular functions” among Ubltargets, assessed by over-representation of Gene Ontology (GO) terms forthe targets of each Ubl and a detailed breakdown of the subset of 189kinases in the network, and their Uhl specificities, showing anextensive crosstalk between kinases and different Ubl modifiers.

FIG. 7 shows an enrichment analyses of “biological processes” among Ubltargets.

FIG. 8 shows the validation of in vitro SUMOylation of known andpredicted kinases. Each reaction was performed by adding a S35-labeledsubstrate into a test tube containing E1, E2, ATP and either recombinantSUMO1, SUMO2 proteins, Reactions were carried out for 2 hours at roomtemperature. Negative control (right lane of each gel) was performedunder the same conditions without adding the E1 enzyme.

FIG. 9 shows that FAT10 targets are involved in cell cycle regulationand mitotic progression. Signal intensity of FAT10ylated protein targetswas measured under nocodazole arrest and upon release from mitoticarrest. Four different replicate spots for each substrate under the twoconditions were compared using ANOVA and the resulting p-valuesindicating the significant change in Ubl modification were plotted(ascending order) for each Uhl separately. The two dotted lines indicatep-value cutoff levels of 0.1 and 0.05 as seen by the orange and brownlines. The y-axis denoted the cumulative number of target proteins thatshowed the stated significance in differential reactivity. Duplicatespots of a FAT10 modified substrate under the two conditions, showingdifferences in reactivity.

FIG. 10 shows the effect of different conditions on FAT10ylation signal.FAT10 signal intensity values of all proteins using two differentantibodies x-axis: peptide epitope. BioMol; y-axis: whole proteinepitope, BioMol). FAT10 signal intensity values of all proteins on thearray with and without washes (3×) with 0.5% SDS.

FIG. 11 shows FAT10 targets that mapped onto a the known interactionnetwork for cell cycle regulation.

FIG. 12 shows a subset of proteins that are differentially modified byFAT10 described to have either a mitotic or death phenotype by RNAinterference as reported in the “mitocheck” database.

FIG. 13 shows the cellular localization of Ube2Z: and FAT10 ininterphase and mitotic cells. Immunofluorescence using either anti-Ube2Zand anti-FAT10 antibodies was done in order to detect their signal inthe cell both in mitosis and interphase. A representative cell of morethan 30 cells is given.

FIG. 14 shows that inhibition of the FAT10 pathway using RNAinterference leads to mitotic arrest. HeLa cells were synchronized usingdouble-thymidine block and released into fresh medium for 15 hours tofollow cell cycle progression. Aliquots were taken for Western blotanalysis of Ube2z, FAT10, Securin and Actin protein levels at theindicated time points. In additions, samples were taken at the same timefor FACS analysis in order to follow cell cycle progression aftersynchronization.

FIG. 15 shows that Ube2Z is stabilized in cells arrested withnocodazole. cells were synchronized by double thymidine block andreleased into medium containing nocodazole. Samples were aliquoted atthe indicated time points and analyzed using propidiume iodide stainingby FACS in order to determined cell cycle stage. In addition, sampleswere collected in the same time points for protein analysis of theindicated proteins using SDS-PAGE and western blot,

FIG. 16 shows that inhibition of ube2z, the FAT10-conjugating enzyme orof FAT10 leads to prolonged mitotic arrest that is followed byapoptosis. HeLa cells transfected with siRNA to ube2z or FAT10 exhibitedprolonged arrest in mitosis when compared to control treated cells. Thearrest in mitosis led to cell death as can be seen by the reduced numberof cells in the Ube2z or FAT10 siRNA treated cells. Three differentexamples are given for each condition.

FIG. 17 shows that inhibition of the FAT10 pathway using RNAinterference leads to mitotic arrest. The duration of mitosis wasquantified from time lapse movies. Cells treated with siRNA for Ube2z orFAT10 spent a significantly longer time in mitosis (p<0.05) whencompared to cells treated with control siRNA. Error bars depict mean andstandard deviation.

FIG. 18 shows that inhibition of the FAT pathway using RNA interferenceleads to mitotic arrest and cell death. A representative cell (n>30 percondition) undergoing mitosis for each of the different condition(siFAT10, siUbe2z and siControl) is presented. Quantitation of thepercentage of cells in interphase (left), mitosis (middle) andpercentage of dead cells (right) in each of the conditions during thecourse of the experiment.

FIG. 19 shows the nucleic acid sequence and the amino acid sequence ofhuman FAT10 (SEQ ID NO: 1 and 2, respectively).

FIG. 20 shows the nucleic acid sequences that encode exemplary FAT10substrates (SEQ ID NO: 3-465, respectively).

FIG. 21 shows tumor growth rate of B16 melanoma tumor cells in FAT10 KO(Squares) and FAT10 wt (Diamonds) mice. 1×10⁵ cells were injected intoC57BL/6 mice and tumor volume (mm³) was assessed every other day.

FIG. 22 shows FAT10 gene expression in immune and cancer cells under thestated conditions. Percentile refers to the relative level of expressionof FAT10 versus the most highly expressed genes in that cell type andcondition.

DETAILED DESCRIPTION

General

Provided herein are compositions and methods for the inhibition ofmitosis, the inhibition of cellular proliferation, the induction ofapoptosis, the treatment of cancer, the treatment of immune disordersand/or the identification of novel therapeutic agents.

As described herein, analysis of Ubiquitin-like protein (Ubl)modification profiles upon cellular release from nocodazole arrest forUbiquitin, SUMO1, SUMO⅔, NEDD8, UFM1, FAT10 and ISG15 showed that allbut two Ubl pathways altered at least some of their targets duringmitosis, indicating that post-transcriptional modification by Ubls playsan important role in mitotic regulation. Among the Ubls investigated,FAT10 exhibited the most dramatic changes in signal intensity and numberof differentially-modified proteins. Although FAT10 itself wasidentified more than a decade ago, little is known about what itregulates. The experiments described herein identified a number of FAT10substrates (e.g., those listed in FIGS. 3 and 10).

As described herein, the reactivity pattern of FAT10 was strikinglydifferent from that of ubiquitin during the metaphase-anaphasetransition. While the poly-ubiquitylation signal increases strongly, theFAT10 signal decreased for 76 out of 106 targets. In support of theimportance of FAT10 in mitosis, inhibiting the FAT10 pathway byknocking-down FAT10 or its E2-conjugating enzyme (Ube2z) resulted in aclear prolongation of the mitotic arrest, followed by cell death. Whatis more, a significant fraction of the FAT10 pathway members identifiedin the studies described herein are important regulators of immunefunction and/or play a role in tumorigenesis and cancer pathogenesis.Examples of FAT10 pathway members that have an immune function areprovided in Table 1. Thus, FAT10 is an attractive target for mitoticinhibition and the treatment of cancer and immune disorders.

TABLE 1 Exemplary FAT10 pathway members with immune function. GeneSymbol Gene Function Protein Seq. ELK3 T cell activation NP_005221.2 GALalpha-beta T cell activation NP_057057.2 IGF2 Immune response-regulatingcell NP_000603.1 surface receptor signaling pathway ABCF1 positiveregulation of immune NP_001020262.1 system process POLR3C regulation ofimmune NP_006459.3 effector process STAT6 regulation of lymphocyteNP_001171549.1 activation AGER regulation of leukocyte activationNP_001127.1 KIAA1715 negative regulation of NP_085153.1 lymphocyteactivation SFXN1 negative regulation of immune NP_073591.2 systemprocess SDC1 response to wounding NP_001006947.1 HP defense responseNP_001119574.1 TRAT1 inflammatory response NP_057472.2 CLEC7A regulationof lymphocyte NP_072092.2 activation HPR T cell activation NP_066275.3RPS6KB1 alpha-beta T cell activation NP_003152.1 SMAD3 regulation ofleukocyte activation NP_001138574.1 ADORA2A positive regulation ofimmune NP_000666.2 system process LAT regulation of immuneNP_001014987.1 effector process IL15 regulation of lymphocyteNP_000576.1 proliferation MICB positive regulation of B NP_005922.2 cellactivation LIG3 positive regulation of NP_002302.2 immune responseSPAG11B regulation of alpha-beta NP_057596.1 T cell activation HSH2Dpositive regulation of NP_116244.1 lymphocyte activation MMP9 immunesystem development NP_004985.2 SPAG11A lymphocyte differentiationNP_001075021.2 GATA3 T cell activation NP_002042.1 S100A12 inflammatoryresponse NP_005612.1 VEGFA T cell activation NP_001020537.2 CCR9 immuneresponse NP_001243298.1 HDAC4 B cell activation NP_006028.2 DHX58 innateimmune response NP_077024.2 IL32 immune response NP_001012649.1 FKBP1B Tcell proliferation NP_004107.1 INS alpha-beta T cell activationNP_000198.1 IL9 immune response NP_000581.1 PRKCQ positive regulation ofNP_001229342.1 T cell activation NMI inflammatory response NP_004679.2BNIP3L defense response to virus NP_004322.1 SYK B cell receptorsignaling pathway NP_001128524.1 CXorf9 positive regulation of BNP_061863.1 cell proliferation VSTM3 negative regulation of NP_776160.2T cell activation IFNW1 defense response NP_002168.1 DEFB4 immuneresponse NP_004933.1 PIK3R1 T cell receptor signaling pathwayNP_001229395.1 RSAD2 defense response to virus NP_542388.2

Thus, in certain embodiments described herein are methods of inhibitingmitosis, inhibiting proliferation and/or inducing apoptosis in a cell bycontacting the cell with an agent that inhibits the FAT10 pathway, suchas an agent that inhibits FAT10 expression and/or activity. In someembodiments, described herein are methods of treating cancer and/orimmune disorders through the inhibition of the FAT10 pathway. In someembodiments described herein are methods of identifying mitoticinhibitors and/or potential cancer or immune disorder therapeutics byidentifying agents that inhibit the FAT10 pathway, such as agents thatinhibit FAT10 expression and/or activity.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “administering” means providing apharmaceutical agent or composition to a subject, and includes, but isnot limited to, administering by a medical professional andself-administering.

The term “agent” is used herein to denote a chemical compound, a smallmolecule, a mixture of chemical compounds, or a biologicalmacromolecule. Agents may be identified as having a particular activityby screening assays described herein below. The activity of such agentsmay render them suitable as a “therapeutic agent” which is abiologically, physiologically, or pharmacologically active substance (orsubstances) that acts locally or systemically in a subject.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing.

As used herein, the term “cancer” includes, but is not limited to, solidtumors and blood borne tumors. The term cancer includes diseases of theskin, tissues, organs, bone, cartilage, blood and vessels. The term“cancer” further encompasses primary and metastatic cancers.

The term “control” includes any portion of an experimental systemdesigned to demonstrate that the factor being tested is responsible forthe observed effect, and is therefore useful to isolate and quantify theeffect of one variable on a system.

The term “FAT10 inhibitor” or “agent that inhibits FAT10” refers to anagent that decreases the level of FAT10 protein and/or decreases atleast one activity of a FAT10 protein. In an exemplary embodiment, aFAT10-inhibing compound may decrease at least one biological activity ofa FAT10 protein by at least about 10%, 25%, 50%, 75%, 100%, or more.Exemplary biological activities of FAT10 proteins include the formationof a conjugate between of FAT10 protein to FAT10 substrates (e.g., thoseencoded by SEQ ID NO: 3-465) and the proteasome-mediated degradation ofFAT10 substrates.

The term “FAT10 pathway” refers to the network of interacting proteinsregulated by FAT10 and proteins that regulate FAT10. Exemplary proteinsthat are components of the FAT10 pathway are provided in FIGS. 3 and 20.Members of the FAT10 pathway that regulate FAT10 activity include the Elligase Uba6 and the E2 ligase Ube2z.

As used herein, the term “immune cell” refers to the cells that make upthe innate and the adaptive immune system. Exemplary immune cellsinclude T cells, B cells, macrophages, dendritic cells, natural killercells, monocytes, neutrophils, eosinophils, basophils and mast cells.

As used herein, the term “immune disorder” refers to any disease,disorder or disease symptom caused by an activity of the immune system,including autoimmune diseases, inflammatory diseases and allergies.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified, such as by conjugation with a labeling component. Theterm “recombinant” polynucleotide means a polynucleotide of genomic,cDNA, semisynthetic, or synthetic origin which either does not occur innature or is linked to another polynucleotide in a non-naturalarrangement.

A “patient” or “subject” refers to either a human or a non-human animal.

The term “small molecule” is art-recognized and refers to a compositionwhich has a molecular weight of less than about 2000 amu, or less thanabout 1000 amu, and even less than about 500 amu. Small molecules maybe, for example, nucleic acids, peptides, polypeptides, peptide nucleicacids, peptidomimetics, carbohydrates, lipids or other organic (carboncontaining) or inorganic molecules. Many pharmaceutical companies haveextensive libraries of chemical and/or biological mixtures, oftenfungal, bacterial, or algal extracts, which can be screened with any ofthe assays described herein. The term “small organic molecule” refers toa small molecule that is often identified as being an organic ormedicinal compound, and does not include molecules that are exclusivelynucleic acids, peptides or polypeptides.

The phrases “therapeutically-effective amount” and “effective amount” asused herein means that amount of a compound, material, or compositioncomprising a compound described herein which is effective for producingsome desired therapeutic effect in at least a sub-population of cells inan animal at a reasonable benefit/risk ratio applicable to any medicaltreatment.

“Treating” a disease in a subject or “treating” a subject having adisease refers to subjecting the subject to a pharmaceutical treatment,e.g., the administration of a drug, such that at least one symptom ofthe disease is decreased or prevented from worsening.

FAT10 Proteins and FAT10 Substrate Proteins

As used herein, the term “FAT10” or “FAT10 protein” refers to the small,Ubiquitin-like modifier encoded in the major histocompatibility complexthat is composed of two ubiquitin-like domains and possessing a freeC-terminal diglycine motif, as well as functional domains, fragments(e.g., functional fragments), e.g., fragments of at least 8 amino acids,e.g., at least 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120amino acids, and variants thereof. Exemplary functional fragments ofFAT10 can, for example, form conjugates with FAT10 substrates andthereby regulate the stability and/or function of the FAT10 substrate.Exemplary FAT10 proteins include those having an amino acid sequence ofSEQ ID NO: 2 (provided in FIG. 19). Homologs of FAT10 proteins willshare 60%, 80%, 85%, 90%, 95%, 98%, 99% sequence identity to a knownFAT10 protein and, e.g., form conjugates with FAT10 substrates. Variantsof FAT10 proteins can be produced by standard means, includingsite-directed and random mutagenesis.

In certain embodiments, it may be advantageous to provide homologs ofFAT10 protein that lack certain aspects of FAT10 activity. Such homologsmay function as a modulator that inhibit a subset of the biologicalactivities of naturally-occurring FAT10. For example, addition of twoalanine residues to the C-terminus of FAT10 prevents it from conjugatingwith FAT10 substrates. Thus, antagonistic homologs may be generatedwhich interfere with the ability of the wild-type FAT10 protein toassociate with certain proteins (e.g., proteins that mediate theconjugation to FAT10 substrates).

As used herein, the term “FAT10 substrate” or “FAT10 substrate protein”refers to a protein that forms a conjugate with FAT10 during a naturalbiological process. Exemplary FAT10 substrates include those encoded bythe nucleic acid sequences provided in FIG. 20 and SEQ ID NO: 3-465. Insome embodiments, conjugation of FAT10 to a FAT10 substrate results inthe proteasome-mediated degradation of the FAT10 substrate. FAT10substrates are components of the FAT10 pathway.

In certain embodiments, a protein described herein is further linked toa heterologous polypeptide, e.g., a polypeptide comprising a domainwhich increases its solubility and/or facilitates its purification,identification, detection, and/or structural characterization. A proteindescribed herein may be linked to at least 2, 3, 4, 5, or moreheterologous polypeptides. Polypeptides may be linked to multiple copiesof the same heterologous polypeptide or may be linked to two or moreheterologous polypeptides. The proteins may also include linkersequences between a protein described herein and the fusion domain inorder to facilitate construction of the fusion protein or to optimizeprotein expression or structural constraints of the fusion protein.

In another embodiment, a protein may be modified so that its rate oftraversing the cellular membrane is increased. For example, thepolypeptide may be fused to a second peptide which promotes“transcytosis,” e.g., uptake of the peptide by cells. The peptide may bea portion of the HIV transactivator (TAT) protein, such as the fragmentcorresponding to residues 37-62 or 48-60 of TAT, portions which havebeen observed to be rapidly taken up by a cell in vitro (Green andLoewenstein, (1989) Cell 55:1179-1188). Alternatively, the internalizingpeptide may be derived from the Drosophila antennapedia protein, orhomologs thereof. The 60 amino acid long homeodomain of thehomeo-protein antennapedia has been demonstrated to translocate throughbiological membranes and can facilitate the translocation ofheterologous polypeptides to which it is coupled. Thus, the polypeptidemay be fused to a peptide consisting of about amino acids 42-58 ofDrosophila antennapedia or shorter fragments for transcytosis (Derossiet al. (1996) J Biol Chem 271:18188-18193; Derossi et al. (1994) J BiolChem 269:10444-10450; and Perez et al. (1992) J Cell Sci 102:717-722).The transcytosis polypeptide may also be a non-naturally-occurringmembrane-translocating sequence (MTS), such as the peptide sequencesdisclosed in U.S. Pat. No. 6,248,558.

FAT10 Nucleic Acids and FAT10 Substrate Nucleic Acids

Nucleic acids encoding any of the proteins described herein (e.g. FAT10and FAT10 substrates) are also provided herein. The nucleic acidsequence of human FAT10 is provided in FIG. 19 and SEQ ID NO: 1. Thenucleic acid sequence of exemplary FAT10 substrates are provided in FIG.20 and SEQ ID NO: 3-465. Such a nucleic acid may further be linked to apromoter and/or other regulatory sequences, as further described herein.Exemplary nucleic acids are those that are at least about 80%, 85%, 90%,95%, 98%, 99% or 100% identical to a nucleotide sequence provided hereinor a fragment thereof, such as nucleic acid sequence encoding theprotein fragments described herein. Nucleic acids may also hybridizespecifically, e.g., under stringent hybridization conditions, to anucleic acid described herein or a fragment thereof.

Nucleic acids, e.g., those encoding a protein described above, afunctional homolog thereof, or a nucleic acid intended to inhibit theproduction of a protein of interest (e.g., siRNA, shRNA or antisenseRNA, described in greater detail below) can be delivered to cells inculture, ex vivo, and in vivo. The delivery of nucleic acids can be byany technique known in the art including viral mediated gene transfer,liposome mediated gene transfer, direct injection into a target tissue,organ, or tumor, injection into vasculature which supplies a targettissue or organ.

Polynucleotides can be administered in any suitable formulations knownin the art. These can be as virus particles, as naked DNA, in liposomes,in complexes with polymeric carriers, etc. Polynucleotides can beadministered to the arteries which feed a tissue or tumor.

Nucleic acids can be delivered in any desired vector. These includeviral or non-viral vectors, including adenovirus vectors,adeno-associated virus vectors, retrovirus vectors, lentivirus vectors,and plasmid vectors. Exemplary types of viruses include HSV (herpessimplex virus), AAV (adeno associated virus), HIV (humanimmunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV(murine leukemia virus). Nucleic acids can be administered in anydesired format that provides sufficiently efficient delivery levels,including in virus particles, in liposomes, in nanoparticles, andcomplexed to polymers.

A polynucleotide of interest can also be combined with a condensingagent to form a gene delivery vehicle. The condensing agent may be apolycation, such as polylysine, polyarginine, polyornithine, protamine,spermine, spermidine, and putrescine. Many suitable methods for makingsuch linkages are known in the art.

In an alternative embodiment, a polynucleotide of interest is associatedwith a liposome to form a gene delivery vehicle. Liposomes are small,lipid vesicles comprised of an aqueous compartment enclosed by a lipidbilayer, typically spherical or slightly elongated structures severalhundred Angstroms in diameter. Under appropriate conditions, a liposomecan fuse with the plasma membrane of a cell or with the membrane of anendocytic vesicle within a cell which has internalized the liposome,thereby releasing its contents into the cytoplasm. Prior to interactionwith the surface of a cell, however, the liposome membrane acts as arelatively impermeable barrier which sequesters and protects itscontents, for example, from degradative enzymes. Additionally, because aliposome is a synthetic structure, specially designed liposomes can beproduced which incorporate desirable features. See Stryer, Biochemistry,pp. 236-240, 1975 (W.H. Freeman, San Francisco, Calif.); Szoka et al.,Biochim. Biophys. Acta 600:1, 1980; Bayer et al., Biochim. Biophys.Acta. 550:464, 1979; Rivnay et al., Meth. Enzymol. 149:119, 1987; Wanget al., PROC. NATL. ACAD. SCI. U.S.A. 84: 7851, 1987, Plant et al.,Anal. Biochem. 176:420, 1989, and U.S. Pat. No. 4,762,915. Liposomes canencapsulate a variety of nucleic acid molecules including DNA, RNA,plasmids, and expression constructs comprising growth factorpolynucleotides such those described herein

Liposomal preparations for use in the methods described herein includecationic (positively charged), anionic (negatively charged) and neutralpreparations. Cationic liposomes have been shown to mediateintracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7416, 1987), mRNA (Malone et al., Proc. Natl. Acad.Sci. USA 86:6077-6081, 1989), and purified transcription factors (Debset al., J. Biol. Chem. 265:10189-10192, 1990), in functional form.Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. See also Felgner et al., Proc. Natl. Acad. Sci. USA 91:5148-5152.87, 1994. Other commercially available liposomes includeTransfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationicliposomes can be prepared from readily available materials usingtechniques well known in the art. See, e.g., Szoka et al., Proc. Natl.Acad. Sci. USA 75:4194-4198, 1978; and WO 90/11092 for descriptions ofthe synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

Inhibitors of the FAT10 Pathway

Certain embodiments described herein relate to methods of inhibitingmitosis, inhibiting cellular proliferation, causing apoptosis, treatingcancer and/or treating an immune disorder. These methods involveadministering an agent that inhibits the FAT10 pathway. For example,such agents may inhibit the activity and/or expression of FAT10. Agentswhich may be used to inhibit the FAT10 pathway and/or FAT10 includeproteins, peptides, small molecules and inhibitory RNA molecules, e.g.,siRNA molecules, shRNA, ribozymes, and antisense oligonucleotides.

Any agent that inhibits FAT10 and/or the FAT10 pathway can be used topractice certain methods described herein. Such agents can be thosedescribed herein, those known in the art, or those identified throughscreening assays (e.g. the screening assays described herein).

In some embodiments, assays used to identify agents useful in themethods described herein include a reaction between FAT10 and one ormore assay components. The other components may be, for example, a testagent (e.g. the potential agent), or a combination of a test agent and aFAT10 substrate (e.g. the FAT10 substrates provided in FIGS. 3 and 20).Agents identified via such assays, such as those described herein, maybe useful, for example, for inhibiting mitosis, treating cancer ortreating immune disorders.

Agents useful in the methods described herein may be obtained from anyavailable source, including systematic libraries of natural and/orsynthetic compounds. Agents may also be obtained by any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; peptoid libraries (libraries of molecules havingthe functionalities of peptides, but with a novel, non-peptide backbonewhich are resistant to enzymatic degradation but which neverthelessremain bioactive; see, e.g., Zuckermann et al., 1994, J Med. Chem.37:2678-85); spatially addressable parallel solid phase or solutionphase libraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library andpeptoid library approaches are limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of agents may be presented in solution (e.g., Houghten, 1992,Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84),chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores,(Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992, Proc NatlAcad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990,Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J Mol. Biol.222:301-310; Ladner, supra.).

Agents useful in the methods described herein may be identified, forexample, using assays for screening candidate or test compounds whichinhibit the formation of a conjugate between FAT10 or a biologicallyactive portion thereof and a FAT10 substrate.

In some embodiments, the assay systems used to identify compounds thatmodulate the activity of FAT10 involves preparing a reaction mixturecontaining FAT10 and a FAT10 substrate under conditions and for a timesufficient to allow FAT10 to conjugate to its substrate. For example,such conditions can be established through the use of a concentratedcell extract. Use of such extracts are described, for example, in theexemplification and in Merbl and Kirschner, Proc Natl Acat Sci USA106:2543-2548 (2009), which is hereby incorporated by reference in itsentirety. In some embodiments a tissue sample, such as a tumor sample,is used to establish conditions to facilitate conjugation of FAT10 toits substrate. In some embodiments, the FAT10 and/or the FAT10 substrateis linked, either directly or indirectly, to a detectable moiety (e.g.,a radioactive, fluorescent, luminescent and/or enzymatic moiety) tofacilitate its detection. In order to test an agent for activity, areaction mixture is prepared in the presence of the compound and acontrol reaction mixture is prepared in the absence of the testcompound. The control reaction mixture may also contain a placebo agent.The test compound can be initially included in the reaction mixture, orcan be added at a time subsequent to the addition of FAT10 and itssubstrate. Control reaction mixtures are incubated without the testcompound or with a placebo. The conjugation of the substrate by FAT10 isthen detected. Substrate conjugation can be detected by any method knownin the art including, but not limited to, using anti-FAT10 antibodiesand/or detectably labeled FAT10 and/or substrate to detect the level ofconjugation. Conjugation of the substrate in the control reaction, butless or no such conjugation in the reaction mixture containing the testcompound, indicates that the compound decreases with the activity ofFAT10.

The assay for agents that inhibit the interaction of FAT10 with itsbinding partner may be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either FAT10 or itssubstrate onto a solid phase and detecting conjugates anchored to thesolid phase at the end of the reaction. In homogeneous assays, theentire reaction is carried out in a liquid phase. In either approach,the order of addition of reactants can be varied to obtain differentinformation about the agents being tested. For example, test compoundsthat interfere with the interaction between FAT10 and the bindingpartner (e.g., by competition) can be identified by conducting thereaction in the presence of the test substance, i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with FAT10and its interactive binding partner. Alternatively, test compounds thatdisrupt preformed conjugates can be tested by adding the test compoundto the reaction mixture after conjugates have been formed. The variousformats are briefly described below.

In a heterogeneous assay system, either FAT10 or its substrate isanchored onto a solid surface or matrix, while the other correspondingnon-anchored component may be labeled, either directly or indirectly. Inpractice, microtitre plates are often utilized for this approach. Theanchored species can be immobilized by a number of methods, eithernon-covalent or covalent, that are well known in the art. Non-covalentattachment can often be accomplished simply by coating the solid surfacewith a solution of FAT10 or its substrate and drying. Alternatively, animmobilized antibody specific for the assay component to be anchored canbe used for this purpose.

A homogeneous assay may also be used to identify inhibitors of FAT10.This is typically a reaction, analogous to those mentioned above, whichis conducted in a liquid phase in the presence or absence of the testagent. The formed conjugates are then separated from unconjugatedcomponents, and the amount of conjugate formed is determined. Asmentioned for heterogeneous assay systems, the order of addition ofreactants to the liquid phase can yield information about which testcompounds inhibit conjugate formation and which disrupt preformedconjugates.

In such a homogeneous assay, the reaction products may be separated fromunreacted assay components by any of a number of standard techniques,including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, conjugates of molecules may be separated fromunconjugated molecules through a series of centrifugal steps, due to thedifferent sedimentation equilibria of conjugates based on theirdifferent sizes and densities (see, for example, Rivas, G., and Minton,A.P., Trends Biochem Sci 1993 Aug;18(8):284-7). Standard chromatographictechniques may also be utilized to separate conjugated molecules fromunconjugated ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format, for example, the relativelylarger conjugate may be separated from the relatively smallerunconjugated components. Immunoprecipitation is another common techniqueutilized for the isolation of a protein-protein conjugates from solution(see, e.g., Ausubel et al (eds.), In: Current Protocols in MolecularBiology, J. Wiley & Sons, New York. 1999). In this technique, allproteins binding to an antibody specific to one of the binding moleculesare precipitated from solution by conjugating the antibody to a beadthat may be readily collected by centrifugation or through theapplication of a magnetic field. The bound assay components may bereleased from the beads, and a second immunoprecipitation stepperformed, this time utilizing antibodies specific for thecorrespondingly different interacting assay component. Alternatively,the presence of the second assay component in the immunoprecipitatedfraction can detected directly using a detectable label, for example, adetectable label linked either directly or indirectly to FAT10 or itssubstrate.

In another embodiment, agents useful in the methods described herein maybe identified using assays for screening candidate or test compoundswhich bind to FAT10 or a biologically active portion thereof.Determining the ability of the test agent to directly bind to FAT10 canbe accomplished, for example, by coupling the compound with a detectablelabel such that binding of the compound to FAT10 can be determined bydetecting the labeled compound in a complex. For example, compounds canbe labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly,and the radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, assay components can beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

Modulators of FAT10 expression may also be identified, for example,using methods wherein a cell is contacted with a candidate compound andthe expression of FAT10 mRNA or protein is determined. The level ofexpression of mRNA or protein in the presence of the candidate compoundis compared to the level of expression of mRNA or protein in the absenceof the candidate compound. The candidate compound can then be identifiedas a modulator of FAT10 expression based on this comparison. Forexample, when expression of FAT10 is greater in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of FAT10 mRNA or protein expression.Conversely, when expression of FAT10 is less in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of FAT10 mRNA or protein expression.

Inhibitory Nucleic Acid Molecules

In certain embodiments, inhibitory nucleic acid molecules thatspecifically target FAT10 mRNA or FAT10 pathway component mRNA (e.g.,antisense molecules, siRNA or shRNA molecules, ribozymes or triplexmolecules) are used in methods described herein. Such molecules areuseful, for example, in methods of inhibiting mitosis, inhibitingproliferation, inducing apoptosis, treating cancer and/or treatingimmune disorders.

The inhibitory nucleic acid molecules described herein may be contactedwith a cell or administered to an organism. Alternatively, constructsencoding these may be contacted with or introduced into a cell ororganism. Antisense constructs, antisense oligonucleotides, RNAinterference constructs or siRNA duplex RNA molecules can be used tointerfere with expression of a protein of interest, e.g., a FAT10protein and/or a FAT10 substrate protein. Typically at least 15, 17, 19,or 21 nucleotides of the complement of the FAT10 mRNA sequence aresufficient for an antisense molecule. Typically at least 19, 21, 22, or23 nucleotides of a target sequence are sufficient for an RNAinterference molecule. The RNA interference molecule may have a 2nucleotide 3′ overhang. If the RNA interference molecule is expressed ina cell from a construct, for example from a hairpin molecule or from aninverted repeat of the desired sequence, then the endogenous cellularmachinery will create the overhangs. RNA interference molecules mayinclude DNA residues, as well as RNA residues.

Inhibitory nucleic acid molecules can be prepared by chemical synthesis,in vitro transcription, or digestion of long dsRNA by Rnase III orDicer. These can be introduced into cells by transfection,electroporation, or other methods known in the art. See Hannon, GJ,2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002,The rest is silence. RNA 7: 1509-1521; Hutvagner G et al., RNAi: Natureabhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232;Brummelkamp, 2002, A system for stable expression of short interferingRNAs in mammalian cells. Science 296: 550-553; Lee N S, Dohjima T, BauerG, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expressionof small interfering RNAs targeted against HIV-1 rev transcripts inhuman cells. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K.(2002). U6-promoter-driven siRNAs with four uridine 3′ overhangsefficiently suppress targeted gene expression in mammalian cells. NatureBiotechnol. 20:497-500; Paddison PJ, Caudy AA, Bernstein E, Hannon GJ,and Conklin DS. (2002). Short hairpin RNAs (shRNAs) inducesequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958;Paul CP, Good PD, Winer I, and Engelke DR. (2002). Effective expressionof small interfering RNA in human cells. Nature Biotechnol. 20:505-508;Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, and Shi Y.(2002). A DNA vector-based RNAi technology to suppress gene expressionin mammalian cells. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu J-Y,DeRuiter SL, and Turner DL. (2002). RNA interference by expression ofshort-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl.Acad. Sci. USA 99(9):6047-6052.

In the present methods, an inhibitory nucleic acid molecule or aninhibitory nucleic acid encoding polynucleotide can be administered tothe subject, for example, as naked RNA, in combination with a deliveryreagent, and/or as a nucleic acid comprising sequences that express thesiRNA or shRNA molecules. In some embodiments the nucleic acidcomprising sequences that express the siRNA or shRNA molecules aredelivered within vectors, e.g. plasmid, viral and bacterial vectors. Anynucleic acid delivery method known in the art can be used in the methodsdescribed herein. Suitable delivery reagents include, but are notlimited to, e.g., the Mirus Transit TKO lipophilic reagent; lipofectin;lipofectamine; cellfectin; polycations (e.g., polylysine),atelocollagen, nanoplexes and liposomes. The use of atelocollagen as adelivery vehicle for nucleic acid molecules is described in Minakuchi etal. Nucleic Acids Res., 32(13):e109 (2004); Hanai et al. Ann NY AcadSci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-12(2008); each of which is incorporated herein in their entirety.

In some embodiments of the methods described herein, liposomes are usedto deliver an inhibitory oligonucleotide to a subject. Liposomessuitable for use in the methods described herein can be formed fromstandard vesicle-forming lipids, which generally include neutral ornegatively charged phospholipids and a sterol, such as cholesterol. Theselection of lipids is generally guided by consideration of factors suchas the desired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are herein incorporated byreference.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure.

Opsonization-inhibiting moieties for use in preparing the liposomesdescribed herein are typically large hydrophilic polymers that are boundto the liposome membrane. As used herein, an opsonization inhibitingmoiety is “bound” to a liposome membrane when it is chemically orphysically attached to the membrane, e.g., by the intercalation of alipid-soluble anchor into the membrane itself, or by binding directly toactive groups of membrane lipids. These opsonization-inhibitinghydrophilic polymers form a protective surface layer that significantlydecreases the uptake of the liposomes by the MMS and RES; e.g., asdescribed in U.S. Pat. No. 4,920,016, the entire disclosure of which isherein incorporated by reference.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM1. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginicacid, carrageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups. Preferably, the opsonization-inhibiting moiety is aPEG, PPG, or derivatives thereof. Liposomes modified with PEG orPEG-derivatives are sometimes called “PEGylated liposomes.”

Pharmaceutical Compositions

Pharmaceutical compositions described herein include any inhibitor ofthe FAT10 pathway, such as an inhibitor of FAT10 activity or, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier or vehicle. The pharmaceutical compositions mayfurther include additional agents for the treatment of cancer and/orimmune disorders. Pharmaceutical compositions described herein areuseful for inhibiting mitosis, treating cancer and/or treating immunedisorders.

A pharmaceutical composition described herein is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, intravenous, intradermal,subcutaneous, oral, transdermal (topical), transmucosal, and rectaladministration.

Toxicity and therapeutic efficacy of the agents described herein can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. While compounds that exhibit toxic side effects can be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods described herein, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Appropriate dosage agents depends upon a number of factors within thescope of knowledge of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of the small molecule will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered.

Therapeutic Methods

Provided herein are methods of treatment of diseases and disorders thatcan be improved by disrupting the FAT10 pathway. In some embodiments,described herein, are therapeutic methods of treating cancer, includinga cancerous tumor, comprising administering to a subject, (e.g., asubject in need thereof), an effective amount of an agent that inhibitsFAT10 and/or the FAT10 pathway. In some embodiments, described herein,are therapeutic methods of treating an immune disorder (e.g., anautoimmune disease, an inflammatory disease and/or an allergy),comprising administering to a subject, (e.g., a subject in needthereof), an effective amount of an agent that inhibits FAT10 and/or theFAT10 pathway.

The pharmaceutical compositions described herein can be delivered by anysuitable route of administration, including orally, nasally, as by, forexample, a spray, rectally, intravaginally, parenterally,intracisternally and topically, as by powders, ointments or drops,including buccally and sublingually. In certain embodiments thepharmaceutical compositions are delivered generally (e.g., via oral orparenteral administration). In certain other embodiments thepharmaceutical compositions are delivered locally through directinjection into a tumor by direct injection into the tumor's blood supply(e.g., arterial or venous blood supply).

In certain embodiments, the methods of treatment described hereininclude administering an agent that inhibits FAT10 and/or the FAT10pathway in conjunction with a second therapeutic agent to the subject.For example, when used for treating cancer, such methods may compriseadministering pharmaceutical compositions described herein inconjunction with one or more chemotherapeutic agents and/or scavengercompounds, including chemotherapeutic agents described herein, as wellas other agents known in the art. When used to treat immune disorders,such methods may include administering pharmaceutical compositionsdescribed herein in conjunction with one or more agents useful for thetreatment of immune disorders, such as immunosuppressants or othertherapeutic agents known in the art.

Conjunctive therapy includes sequential, simultaneous and separate, orco-administration of the active compound in a way that the therapeuticeffects of the first agent administered have not entirely disappearedwhen the subsequent agent is administered. In certain embodiments, thesecond agent may be co-formulated with the first agent or be formulatedin a separate pharmaceutical composition.

In some embodiments, the subject pharmaceutical compositions describedherein will incorporate the substance or substances to be delivered inan amount sufficient to deliver to a patient a therapeutically effectiveamount of an incorporated therapeutic agent or other material as part ofa prophylactic or therapeutic treatment. The desired concentration ofthe active compound in the particle will depend on absorption,inactivation, and excretion rates of the drug as well as the deliveryrate of the compound. It is to be noted that dosage values may also varywith the severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions. Typically, dosing will be determinedusing techniques known to one skilled in the art.

In certain embodiments, described herein are therapeutic methods oftreating cancer in a subject in need thereof. A subject in need thereofmay include, for example, a subject who has been diagnosed with a tumor,including a pre-cancerous tumor, a cancer, or a subject who has beentreated, including subjects that have been refractory to the previoustreatment.

The methods described herein may be used to treat any cancerous orpre-cancerous tumor. In certain embodiments, the tumor has increasedexpression of FAT10 protein or mRNA relative to non-tumor tissue (e.g.,a non-tumor tissue of the same tissue type as the tumor). Cancers thatmay treated, prevented or diagnosed by methods and compositionsdescribed herein include, but are not limited to, cancer cells from thebladder, blood, bone, bone marrow, brain, breast, colon, esophagus,gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck,ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition,the cancer may specifically be of the following histological type,though it is not limited to these: neoplasm, malignant; carcinoma;carcinoma, undifferentiated; giant and spindle cell carcinoma; smallcell carcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; and roblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.

In some embodiments, described herein are thereapeutic methods fortreating an immune disorder. Such methods can be used to treat anyimmune disorder, including an autoimmune disease (e.g., Lupus,Scleroderma, hemolytic anemia, vasculitis, type one diabetes, Grave'sdisease, rheumatoid arthritis, multiple sclerosis, Goodpasture'ssyndrome, pernicious anemia and/or myopathy), an inflammatory disease(e.g., acne vulgaris, asthma, celiac disease, chronic prostatitis,glomerulonephritis, inflammatory bowel disease, pelvic inflammatorydisease, reprofusion injury, rheumatoid arthritis, sarcoidosis,transplant rejection, vasculitis and/or interstial cystitis), and/or anallergy (e.g., food allergies, drug allergies and/or environmentalallergies).

All publications, including patents, applications, and GenBank Accessionnumbers mentioned herein are hereby incorporated by reference in theirentirety as if each individual publication or patent was specificallyand individually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXEMPLIFICATION

Materials and Methods

HeLa S3 cells were synchronized in prometaphase by treatment withnocodazole. Cells were incubated in thymidine-containing (2 mM) mediumfor 24 hours. Cells were released into fresh medium for 8 hours,followed by a nocodazole arrest (0.1 μg/mL) for 12 hours. Cells wereharvested, washed with PBS, and processed for extraction.

To deplete FAT10 and Ube2z, Dharmacon siGENOME SMARTpool against FAT10or Ube2z (M-008266-03 and M-008596-02, respectively), were used in allexperiments at a final concentration of 20 nM. As a control DharmaconsiGENOME Non-Targeting siRNA Pool #1 and #2 were used at 20 nM(D-001206-13-05 and D-001206-14-05, respectively). siRNA transfectionwas performed using Oligofectamine (Invitrogen) according to themanufacturer's instructions.

Cells were seeded in glass-bottom plates (MatTek) in CO2-independentmedium (Invitrogen) supplemented with 10 FBS, 100 U/ml penicillin and100 μg/ml streptomycin. For fluorescent time-lapse imaging cells wereseeded in phenol red-free CO2-independent medium (Invitrogen). Imageacquisition was performed using Nikon TE2000 automated invertedmicroscope with a 20 objective enclosed in a humidified incubationchamber maintained at 37° C. Images were collected every 15 minutesusing a motorized stage. Images were viewed and analyzed using MetaMorphsoftware (Molecular Dynamics).

Extracts were prepared as described in Merbl and Kirschner, Proc NatlAcad Sci USA 106:2543-2548 (2009), Rape and Kirschner, Nature432:588-595 (2004) and Storey et al., Biostatistics 8:414-432 (2007),each of which is incorporated by reference in its entirety. andincubated on microarrays as described in Merbl and Kirschner, Proc NatlAcad Sci USA 106:2543-2548 (2009) with the following primary antibodies:polyubiquitin antibody (FK1; Biomol), SUMO⅔ (Cell Signaling), NEDD8(Cell signaling), FAT10 (Enzo life sciences), SUMO1 (Cell signaling),UFM1 (BioMol) and ISG15 (Cell signaling). Antibodies were diluted 1:250and detected using fluorescently-labeled secondary antibodies.

The Degradation assay was performed as described in Williamson et al.,Methods Mol Biol 545:301-312, which is incorporated by reference in itsentirety.

For microarray scanning and data processing, Images were acquired usinga GenePix 4000B scanner and processed as described in in Merbl andKirschner, Proc Natl Acad Sci USA 106:2543-2548 (2009).

For Constructing the Ubl network, data were normalized using thequantile normalization algorithm for each modification separately. Inorder to establish an unbiased method for identifying the reactiveproteins in each Ubl reactivity profile, the signal intensity of eachprotein was plotted as a function of their corresponding RFU and binnedinto 100 bins along the RFU range. The RFU value was determined duringthe quality control procedure for each ‘batch’ of microarray production.Since every protein that is spotted on the array is expressed andpurified with a GST tag, amount of material in each spot can beestimated based on the reactivity value towards a labeled anti-GSTantibody. Thus, using both the signal intensity value and the amount ofprotein in each spot the set of targets for each Ubl was identified.Next, in order to establish an unbiased method for identifying thereactive proteins in each Ubl reactivity profile, the signal intensityof each protein was plotted as a function of their corresponding RFUvalues and binned into 100 bins along the RFU range (see FIG. 1). Themean value and standard deviation (SD) of the signal intensities in eachbin were calculated and the best fit line of each measure was calculatedusing linear least square regression analysis. The distance of the SDfrom the line for each bin was calculated. Based on the complementaryerror function a p-value was assigned for each protein and a thresholdlevel of p<0.01 (after false discovery rate (FDR) correction) was set inorder to identify the reactive targets from each Ubl profile.

The mean value and SD of the signal intensities in each bin werecalculated and the best fit line of each measure was calculated usinglinear least square regression analysis. The distance of the SD from theline for each bin was calculated. Based on the complementary errorfunction:

$\begin{matrix}{{{erfc}(x)} = {1 - {{erf}(x)}}} \\{= {\frac{2}{\pi}{\int_{x}^{\infty}{e^{- t^{2}}{{dt}.}}}}}\end{matrix}\quad$a p-value was assigned for each protein. The error function representsthe probability that the parameter of interest is within a range between−x/σ√2 and x/σ√2, while the complementary error function provides theprobability that the parameter is outside that range. A threshold levelof p<0.01 (after false discovery rate (FDR) correction) was set in orderto select the set of ‘positive’ targets from each Ubl profile. A totalof 1543 target proteins passed the threshold for at least one of themodifications.

To identify Ubl targets that are differentially modified upon mitoticrelease, the reactivity level of each protein was compared in differentconditions using the ANOVA test (4 duplicate spots per condition).Significance was determined based on Storey's pvalue correction.

The in vitro SUMOylation analysis was performed as follows. E1, and E2enzymes were added to a S35-radioactively labeled substrate withrecombinant SUMO1, SUMO2 or SUMO3. The reaction was supplemented withATP and allowed to run in room temperature for 2 hours. As a negativecontrol, the same reaction is performed without the addition of the E1enzyme and ran for the same amount of time (the right lane of each gel).Reactions are stopped by addition of sample buffer containing5%β-mercaptoethanol. To identify modified substrates the samples wereanalyzed by SDS-PAGE and phosphorimaging. The formation of a highermolecular-weight species (ladder) signifies the substrate'smodification.

Example 1 Global Identification of Ubiquitin and Ubl Targets in Mitosis

An assay in which concentrated cell extracts were applied directly tomicroarrays and the modification of a subset of proteins was determinedusing modification-specific antibodies was used to explore the role ofseveral ubiquitin-like modifications (Ubiquitin, SUMO⅔, NEDD8, FAT10,SUMO1, UFM1 and ISG15) in mitotic regulation. For these sevenmodifications, the modification state of thousands of proteins beforeand after release from mitotic arrest were profiled (FIG. 2). Theseextracts promote full checkpoint arrest and APC inhibition and to berelieved of that inhibition by Ubch10. Extracts were applied toduplicate microarrays for each modification under each of twoconditions: ‘arrested’ (blocked in mitosis with nocadazole) and‘released’ (released into anaphase/G1 from that block by Ubch10) for atotal of 28 microarrays. The mean reactivity of each target protein wasthen calculated from four replicate spots (2 spots per array×2replicates).

The subsequent analysis focused on highly reactive proteins, defined ashaving a specific reactivity greater than 2 standard deviations abovethe mean for each Ubl modification (when normalized to the proteinabundance on the chip; see materials and methods and FIG. 1). Allproteins passing these criteria also have a reactivity significantlyhigher than the background reactivity of negative control spots, whichcontain either no protein, or else the GST-tag alone, or bovine serumalbumin. Each Ubl reacted with 158-506 proteins that exhibited Ublreactivity greater than the threshold. 1543 such target proteins highlyreactive to at least one of the Ubls were identified (FIG. 3). Forubiquitin, which is the most investigated protein in this family,approximately 70% of the targets previously identified were confirmedeither in vitro or in vivo as ubiquitylation substrates. Thus, thefalse-negative rate of the assay was low.

Example 2 The Ubl Modification Network

Each reactive protein target's interaction with each of the seven Ublmodifications were characterized (FIG. 3). Some proteins are reactive tojust one Ubl (e.g. Rad23a. which exhibited high reactivity only towardsubiquitin), while others react with multiple Ubls (e.g. Igf1R). Severalexamples of such interactions were previously reported and are shown inFIG. 4A. Among these, a few have been identified only recently.

To identify global patterns of Ubl modifications, the different targetsfor each Ubl were mapped into an “interaction network” (FIG. 4B). Thenetwork consists of seven hubs, corresponding to the Ubls, and multiplenodes representing each one of the target proteins. Edges between thehubs and nodes represent the PTM interactions. The network reveals theinteraction of the different Ubls with their targets, and their degreeof specificity. Most of the Ubl targets (65% of 1543) map just to asingle Ubl (FIG. 4B, proteins assembled at the rim of the graph),whereas the remaining targets (35%) map to at least two Ubls (proteinsin the center of the graph). Thus, most proteins are regulated primarilyby one Ubl, though this varied from Ubl to Ubl. For example, only 20%were unique to SUMO1, whereas 68% of FAT10 targets were unique to FAT10(FIG. 4C).

The network (FIG. 4B) reveals that, a large number of target proteinsinteract with multiple Ubls, with a few (2.6%) interacting with five ormore modifications (nodes in the center of the network). This patternsuggests that there is considerable specificity in Ubl modification.

To characterize the statistical implications of a network of sevenindependent Ubl modifications, the fraction of nodes (protein targets),F_(n), with 1≤n≤7 edges connecting them to hubs corresponding to theseven Ubl modifications were considered. If the different Ublmodifications were independent of each other, then F_(n) shouldcorrespond to a multinomial distribution, where the edges from each ofthe seven hubs is assigned to targets at random. To test whether this isthe case, 5000 random permutations of the network that preserved thenumber of edges from each hub were generated, and the resulting edgedistribution F_(n) to the empirical data were compared (FIG. 5). Theanalysis shows that the number of unique targets F₁ (proteins targetedby only one Ubl), was higher than would be expected by chance (65%observed versus 52±1.4% expected), while the fraction F₂ ofdoubly-modified targets was much lower than expected by chance (19%observed versus 34±1% expected). Therefore, on average, the subset ofdoubly-modified targeted proteins (F₂) appear to be anti-correlated: aprotein modified by one Ubl is less likely to be targeted by another.

The low abundance of doubly-modified targets suggests that certain Ublcombinations might be permitted while the majority is not (FIG. 5). Apair-wise correlation analysis of Ubl modifications confirmed that,indeed, most of the possible double Ubl interactions are stronglysuppressed. However, certain Ubl combinations are over-represented inthe correlation map, indicating that certain Ubls often co-target thesame substrates. For example, there was high correlation between SUMO1and SUMO⅔ targets (R=0.58), as well as between UFM1 and FAT10modifications (R=0.63).

The enrichment of the network for exclusive Ubl targets (F₁) can bestudied by comparing specific Ubl reactivity combinations to thepredictions of randomized permutations. Since 7 Ubls were profiled, 127(2^7−1) possible “reactivity states” for different Ublscombinations/patterns were identified. For single-targeted proteins,only FAT10 and UFM1 exhibited a higher frequency of exclusive targetscompared to random: 324 unique FAT10 targets were observed when 161 wereexpected, and 202 UFM1 targets were observed compared to 108 expected,suggesting that the functions of the FAT10 and UFM1 systems are largelyinsulated from the activity of other Ubls. The other Ubls (e.g.ubiquitin, SUMO1, SUMO⅔) exhibited the same frequency of exclusivetargets as would be expected by chance, suggesting that these parallelpathways evolved independently. Thus, the over-representation of uniquesubstrates is largely dominated by UFM1 and FAT10.

Example 3 Cellular and Functional Classification of Ubl Targets

To examine whether the different Ubl pathways might be targeted tospecific categories of proteins, or associated with distinct classes ofbiological processes, over-represented Gene Ontology (GO) terms for Ubltarget protein annotations were identified using the Panther database(http://www.pantherdb.org). For each Ubl, the enrichment for GO termsidentified with its substrates was determined. Functional terms werescored based on their enrichment compared to the complete list of targetproteins with significant enrichment determined by p-value<0.1(corrected for false discovery rate). By comparing the targets of eachUbl to the list of reactive proteins only, false-positive enrichmentsthat could arise from biases in the representation of protein subsets onthe chip were limited. FIG. 6 presents the molecular functions (thefunction that the protein performs on its direct molecular targets)related to the targets of each Ubl, while FIG. 7 presents the biologicalprocesses (the systems to which the protein contributes). Each columnrepresents one Ubl (with enriched terms shaded).

The only over-represented categories in the cell cycle were ‘mitosis’and ‘cytokinesis’, indicating that the assay preferentially identifiedproteins involved in mitotic regulation. Known biological functions ofUbiquitin and SUMO were enriched (FIG. 6). For example, ubiquitylationtargets are the only ones enriched for the ‘Ubiquitin ligase activity’term, with numerous targets categorized as E3 ligases or ‘ring finger’proteins. In addition, SUMO1 and SUMO 2/3 targets are uniquely enrichedfor transcriptional-related terms and DNA binding corresponding to theirrole in transcriptional regulation.

The assay revealed a class of ‘translation initiation factors’ targetedby the ISG15 modification (FIG. 3). UFM1 targets were enriched intransmembrane transporters, ion channels and cytokine receptors, andFAT10 targets were enriched for SNAP receptors, proteins related toextracellular matrix activity and DNA helicases.

Kinases were enriched in the set of Ubl targets (FIG. 6). Specifically,SUMO1 and SUMO⅔ together targeted 181 of the 189 kinases (FIGS. 6) thatwere reactive in the assay. Among these, 20 known mitotic kinases werefound. The SUMOylation of Polo-like kinase 1 (Plk1) and Cyclin dependentkinase-1 (Cdk1) as well as other kinases were confirmed in vitro (FIG.8).

Many of the Ubls were implicated in a common set of biological processes(FIG. 7) that include cell cycle regulation, apoptosis, angiogenesis,cell adhesion and embryonic development. FAT10 and UFM1 are theexceptions: FAT10 targets were over-represented in ‘antigen processingand presentation via MHC class II’ and ‘cellular calcium homeostasis’,while UFM1 targets were implicated in pathways classified as‘endocytosis’, ‘hemopoeisis’, ‘neurotransmitter secretion’ and ‘lipidtransport’. Both FAT10 and UFM1 also had a significant number of targetsin the mitosis/cytokinesis pathways. For example, 27 of the proteinsmodified by FAT10 were cell cycle regulators, 19 of which weremitosis-related.

Example 4 Differential Regulation of FAT10 Upon Release from MitoticArrest

To examine prometaphase arrest to anaphase/G1 under conditions thatminimized variation in the extracts, a nocodazole-arrested HeLa cellextract (checkpoint arrested; denoted ‘arrested’) was compared with thesame extract supplemented with the E2 enzyme, Ubch10, which relievesmitotic arrest (denoted as ‘released’) and drives the cell into anaphaseand Gl. The reactivity level of each protein towards each of the Ublswas calculated for these two conditions, using two microarrays percondition. An ANOVA test performed for the reactivity of each proteinunder these conditions (whose p-values were corrected using Storey'sfalse discovery rate method) showed that the reactivity for bothubiquitin and FAT10 had the most significant changes (q-value<0.1) uponrelease from the arrested into the released state (FIG. 9).

FIG. 9 depicts examples of FAT10 reactivity levels for several proteinsunder the two conditions. Most of these statistically significantchanges in FAT10 level represented dramatic decreases of two to threeorders of magnitude. This effect could not be explained by thevariability of the assay and persisted if different antibodies were used(FIG. 10, top) or more stringent washing conditions were used (FIG. 10,bottom). Thus, 106 proteins showed a significant difference, either anincrease in FAT10ylation (30 proteins) or a decrease (76 proteins),between the arrested and released conditions in FAT10 reactivitypatterns (q<0.002).

Example 5 FAT10 is Involved in Cell Cycle Regulation and MitoticProgression

Among the proteins that showed a change in FAT10 signal intensity werethose that mapped onto a known interaction network related to cell cycleregulation (e.g. securin, cull, septin 6 and cdk3; see FIG. 11),indicating that the FAT10 pathway is important for mitosis. Indeed, whenthis list of FAT10 changes were compared to a database of the phenotypicoutcomes of a genome-wide RNA interference screen, it was revealed that8 of the candidates have a mitotic phenotype (delay) upon knockdown and2 additional genes to have a ‘death phenotype’ (FIG. 12).

The only known E2-conjugation enzyme for the FAT10 pathway is Ube2z, aprotein that is highly conserved in vertebrates and is expressed at highlevels in various human cancer cell lines from the NCI-60 collection.Using immunofluorescence, it was determined that Ube2z is ubiquitouslyexpressed both in interphase and mitosis in HeLa cells (FIG. 13). Tomeasure levels of Ube2z in mitosis, cells were synchronized usingdouble-thymidine block and released them into fresh media. Ube2z levelswere high during G2/M and dropped precipitously in mitosis with similartiming to that of securin degradation (FIG. 14). When the cells werereleased from the thymidine block into nocodazole, Ube2z was completelystable even 15 hours after the release (FIG. 15). The timing of thedegradation of Ube2z indicates that it may be required for the mitoticcheckpoint and that its degradation may be regulated in an APC dependentmanner.

To determine if Ube2z is an APC substrate, the ability of the specificAPC inhibitory protein, Emil, to inhibit Ube2z degradation in mitoticextracts was tested. In the absence of Emil, Ube2z levels droppedgradually within 90 minutes (80% reduction). In the presence of Emil thedegradation of Ube2z is completely blocked, indicating that Ube2z levelsare regulated by APC.

To look at the regulatory role of FAT10 in mitosis, the effects ofinhibiting FAT10ylation via either a knockdown of FAT10, or by knockdownof its E2-conjugating enzyme, Ube2z were examined. HeLa cells weretransfected with either siRNA against FAT10 or Ube2z, or with controlsiRNA and allowed the cells to grow for 72 hours. In both knockdowns butnot in the control there was a substantial increase in the duration ofmitosis, eventually leading to cell death (FIG. 16). When the averageduration in mitosis was quantified (FIG. 17), it was found that cellsstayed in mitosis at least twice as long, on average than controls(5.93±1.96 and 8.48±2.58 hours for Ube2z and FAT10, respectively), whencompared to cells transfected with control siRNA (2±0.8 hours). Thus,both the inhibition of FAT10 and the inhibition of Ube2z extendedmitotic arrest triggered cell death (FIG. 18).

Example 6 Loss of FAT10 Function Inhibits Cancer Progression

The role of FAT10 in tumor development was investigated using a mousetumor model for subcutaneous melanoma. The subcutaneous model is widelyused for the evaluation of therapy in many tumor models, including B16melanoma. C57BL/6 wild-type (wt; n=7) mice or FAT10 knockout (KO; n=8)mice (C57BL/6 background) were injected subcutaneously with B16 melaomatumor cells. A dose of 1×10⁵ cells/mouse, which is 1.5 to 2 times theminimal tumorigenic dose in normal C57BL/6 mice, was used for bothcontrol and KO mice. Upon subcutaneous injection, B16 form a palpabletumor in 5 to 10 days and grew to a 1 cm tumor in 14 to 21 days. Tumorgrowth was manually inspected and measured every other day and recordedaccordingly.

As depicted in FIG. 21, deficiency of FAT10 inhibited the ability oftumors to grow in mice and resulted in a significantly slower rate oftumor growth in the KO mice when compared to the wt. Since the the tumorcells that were injected to both the control and KO mice were identical,the difference in tumor growth observed cannot be the result of adifference in the tumor cells themselves, but rather reflect the abilityof the environment to support tumor cell growth.

Inhibition of tumor growth in FAT10 deficient mice may be the result ofdifference in immune function. Analysis of cDNA microarray datasuggested that FAT10 is expressed in several types of immune cells andconditions (FIG. 22). Indeed, increased FAT10 mRNA levels were detectedby qPCR upon activation of NK, dendritic cells and macrophages that wereisolated from wild-type C57BL/6 mice and activated in vitro.

We claim:
 1. A method of treating melanoma in a subject comprisingadministering to the subject an inhibitory nucleic acid specific for anmRNA that encodes FAT10.
 2. The method of claim 1, wherein theinhibitory nucleic acid is selected from the group consisting of siRNA,shRNA, and an antisense RNA molecule, or a nucleic acid that encodes amolecule selected from the group consisting of siRNA, shRNA, and/or anantisense RNA molecule.
 3. The method of claim 1, wherein the inhibitorynucleic acid inhibits the formation of a conjugate between FAT10 and aFAT10 substrate.
 4. The method of claim 3, wherein the FAT10 substrateis encoded by a nucleic acid having a sequence of SEQ ID NO:
 40. 5. Amethod of treating breast cancer in a subject comprising administeringto the subject an inhibitory nucleic acid specific for an mRNA thatencodes FAT10.
 6. A method of treating cervical cancer in a subjectcomprising administering to the subject an inhibitory nucleic acidspecific for an mRNA that encodes FAT10.
 7. The method of claim 5,wherein the inhibitory nucleic acid is selected from the groupconsisting of siRNA, shRNA, and an antisense RNA molecule, or a nucleicacid that encodes a molecule selected from the group consisting ofsiRNA, shRNA, and/or an antisense RNA molecule.
 8. The method of claim5, wherein the inhibitory nucleic acid inhibits the formation of aconjugate between FAT10 and a FAT10 substrate.
 9. The method of claim 8,wherein the FAT10 substrate is encoded by a nucleic acid having asequence of SEQ ID NO:
 40. 10. The method of claim 6, wherein theinhibitory nucleic acid is selected from the group consisting of siRNA,shRNA, and an antisense RNA molecule, or a nucleic acid that encodes amolecule selected from the group consisting of siRNA, shRNA, and/or anantisense RNA molecule.
 11. The method of claim 6, wherein theinhibitory nucleic acid inhibits the formation of a conjugate betweenFAT10 and a FAT10 substrate.
 12. The method of claim 11, wherein theFAT10 substrate is encoded by a nucleic acid having a sequence of SEQ IDNO: 40.